The major objective of this proposal is to develop viral vectors based on human foamy virus (HFV) for use in gene therapy, especially applications requiring the genetic modification of hematopoietic stem cells. HFV is a retrovirus that offers several potent advantages as a vector system, including a wide host range, improved transduction of non-dividing cells, large packaging capacity, resistance to serum inactivation, and lack of pathogenicity. These properties, along with the ability of HFV vectors to efficiently transduce hematopoietic cells, suggests that they may overcome some of the barriers that have inhibited stem cell transduction by other types of viral vectors. Recently, methods for the production of high titer, helper- free HFV vector stocks have been developed, making possible definitive preclinical studies with this novel vector system. In this proposal HFV vectors with an expanded packaging capacity and improved safety features will be constructed by removing all non-essential cis-actin viral sequences. HFV packaging constructs will be designed that optimize the expression of essential trans-acting viral functions and these will be incorporated into stable packaging cell lines. These improved HFV vectors will be used to transduce primary hematopoietic progenitors from mice, non-human primates and humans. Irradiated mice will be transplanted with congenic marrow cells transduced ex vivo, and the transduction rates of long-term repopulating stem cells will be determined by transgene expression assays and an analysis of vector pro-virus copy numbers. Similar autologous transplantation experiments will be performed in baboons as a non-human primate model, and human hematopoietic cells will be investigated by transplanting transduced human cells into immunodeficient NOD/SCID mice. A variety of experimental variables will be evaluated, including a range of vector MOIs, different reporter genes (alkaline phosphatase, neo, and GFP), several internal promoters, and various transduction conditions, with the goal of developing methods that result in high level, long-term transgene expression in the reconstituted hematopoietic system. Globin expression constructs will be incorporated into optimized HFV vectors. It is hoped that these experiments will lead to effective genetic tretments for the many diseases that require efficient gene transfer into hematopoietic stem cells for therapeutic effect, including the hemoglobinopathies.